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Construction and characterization of an azurin analog for the purple copper site in cytochrome c oxidase

Hay, Michael and Richards, John H. and Lu, Yi (1996) Construction and characterization of an azurin analog for the purple copper site in cytochrome c oxidase. Proceedings of the National Academy of Sciences of the United States of America, 93 (1). pp. 461-464. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:HAYpnas96

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Abstract

A protein analog of a purple copper center has been constructed from a recombinant blue copper protein (Pseudomonas aeruginosa azurin) by replacing the loop containing the three ligands to the blue copper center with the corresponding loop of the CuA center in cytochrome c oxidase (COX) from Paracoccus denitrificans. The electronic absorption in the UV and visible region (UV-vis) and electron paramagnetic resonance (EPR) spectra of this analog are remarkably similar to those of the native CuA center in COX from Paracoccus denitrificans. The above spectra can be obtained upon addition of a mixture of Cu2+ and Cu+. Addition of Cu2+ only results in a UV-vis spectrum consisting of absorptions from both a purple copper center and a blue copper center. This spectrum can be converted to the spectrum of a pure purple copper by a prolonged incubation in the air, or by addition of excess ascorbate. The azurin mutant reported here is an example of an engineered purple copper center with the A480/A530 ratio greater than 1 and with no detectable hyperfines, similar to those of the CuA sites in COX of bovine heart and of Paracoccus denitrificans.


Item Type:Article
Additional Information:© 1996 by the National Academy of Sciences. Communicated by Harry B. Gray, California Institute of Technology, Pasadena, CA, September 12, 1995. We thank Sung Syn for technical assistance, Hong-Chang Liang and Prof. Patricia Shapley for providing purified CuCl, Dr. Alexej Smirnov and Prof. Linn Belford for help in collecting EPR spectrum, Profs. Robert B. Gennis, Stephen G. Sligar, Thomas B. Rauchfuss, and Kenneth S. Suslick and their laboratory members for using the equipment and facilities while the laboratory of Y.L. was being set up, Prof. Bo G. Malmstrom for communication of results before publication, and Prof. Harry B. Gray for helpful discussions. The research is supported by the Department of Chemistry at the University of Illinois at Urbana-Champaign (to Y.L.), the National Science Foundation (Career Award CHE 95-02421 to Y.L.), and the National Institutes of Health (GM16424 to J.H.R.). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Subject Keywords:blue copper; site-directed mutagenesis
Record Number:CaltechAUTHORS:HAYpnas96
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:HAYpnas96
Alternative URL:http://dx.doi.org/10.1073/pnas.93.1.461
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7356
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:05 Feb 2007
Last Modified:14 Nov 2014 19:19

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